Application of electrospray ionization product ion spectra for identification with atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry - a case study with seized drugs.

Product ion spectra obtained with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) were applied to the identification of seized drug samples from atmospheric pressure matrix-assisted laser desorption/ionization product ion spectra (AP-MALDI-MS/MS spectra). Data acquisition was performed in the information-dependent acquisition (IDA) mode, and the substance identification was based on a spectral library previously created with LC-ESI/MS/MS using protonated molecules as precursor ions. A total of 39 seized drug samples were analyzed with both AP-MALDI and LC-ESI techniques using the same triple-quadrupole instrument (AB Sciex 4000QTRAP). The study shows that ESI-MS/MS spectra can be directly utilized in AP-MALDI-MS/MS measurements as the average fit and purity score percentages with AP-MALDI were 90% and 85%, respectively, being similar to or even better than those obtained with the reference LC/ESI-MS/MS method. This fact enables the possibility to use large ESI spectral libraries, not only to ESI analyses but also to analyses with other ionization techniques which produce protonated molecules as the base peak. The data obtained shows that spectral library search works also for analytical techniques which produce multi-component mass spectra, such as AP-MALDI, unless isobaric compounds are encountered. The spectral library search was successfully applied to rapid identification of confiscated drugs by AP-MALDI-IDA-MS/MS.

Combined drug screening and confirmation by liquid chromatography time-of-flight mass spectrometry with reverse database search.

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1-3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10-100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

Measuring insulin in human vitreous humour using LC-MS/MS.

Besides its particular importance as a widely used therapeutic agent, insulin (and its synthetic derivatives) has been suspected, purported, and proven to be a lethal weapon in numerous cases of attempted or successful homicide and suicide. In addition to blood and urine as common matrices for clinical diagnosis and post-mortem analysis, vitreous humour has gained considerable attention in autopsy and follow-up investigations due to its ability to provide valuable information on cause and time of death. However, post-mortem insulin analyses using such specimens have been rare due to the limited penetration of peptide hormones into the vitreous body, and immunoassays were exclusively employed in those studies. In the present communication, the determination of insulin(s) from vitreous humour by means of immunopurification combined with ultrahigh performance liquid chromatography--high resolution/high accuracy (tandem) mass spectrometry is reported. Exploiting the constantly increasing sensitivity and robustness of modern mass-spectrometry-based instruments, the option to identify insulin in post-mortem vitreous samples is demonstrated with a specimen collected from a non-diabetic victim who died from an insulin overdose. This communication represents the first successful mass-spectrometry-based analysis of post-mortem material related to an insulin poisoning case.

Matrix effect in the analysis of drugs of abuse from urine with desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS).

We have studied the matrix effect within direct analysis of benzodiazepines and opioids from urine with desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). The urine matrix was found to affect the ionization mechanism of the opioids in DAPPI-MS favoring proton transfer over charge exchange reaction. The sensitivity for the drugs in solvent matrix was at the same level with DESI-MS and DAPPI-MS (LODs 0.05-6 µg mL(-1)) but the decrease in sensitivity due to the urine matrix was higher with DESI (typically 20-160-fold) than with DAPPI (typically 2-15-fold) indicating better matrix tolerance of DAPPI over DESI. Also in MS/MS mode, DAPPI provided better sensitivity than DESI for the drugs in urine. The feasibility of DAPPI-MS/MS was then studied in screening the same drugs from five authentic, forensic post mortem urine samples. A reference measurement with gas chromatography-mass spectrometry (GC-MS) (including pretreatment) revealed 16 findings from the samples, whereas with DAPPI-MS/MS after sample pretreatment, 15 findings were made. Sample pretreatment was found necessary, since only eight findings were made from the same samples untreated.

Minimum proton affinity for efficient ionization with atmospheric pressure desorption/ionization on silicon mass spectrometry.

We performed a systematic study using a set of compounds with different proton affinities (PAs) on the ionization in atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS). The compounds studied included various aromatic molecules of different sizes. The PAs of these compounds were calculated using ab initio and hybrid density functional theory calculations at the B3LYP/6-31G(d) level of theory. We observed that only compounds with relatively high PAs above a threshold value of 920-950 kJ/mol were efficiently ionized as protonated molecules under AP-DIOS conditions and produced very clean mass spectra.

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry.

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.

Gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry.

An atmospheric pressure chemical ionization (APCI) microchip is presented for combining a gas chromatograph (GC) to a mass spectrometer (MS). The chip includes capillary insertion channel, stopper, vaporizer channel, nozzle and nebulizer gas inlet fabricated on the silicon wafer, and a platinum heater sputtered on a glass wafer. These two wafers are joined by anodic bonding creating a two-dimensional version of an APCI microchip. The sample from GC is directed via heated transfer line capillary to the vaporizer channel of the APCI chip. The etched nozzle forms narrow sample plume, which is ionized by an external corona discharge needle, and the ions are analyzed by a mass spectrometer. The GC-microchip APCI-MS combination provides an efficient method for qualitative and quantitative analysis. The spectra produced by microchip APCI show intensive protonated molecule and some fragmentation products as in classical chemical ionization for structure elucidation. In quantitative analysis the GC-microchip APCI-MS showed good linearity (r(2) = 0.9989) and repeatability (relative standard deviation 4.4%). The limits of detection with signal-to-noise ratio of three were between 0.5 and 2 micromol/L with MS mode using selected ion monitoring and 0.05 micromol/L with MS/MS using multiple reaction monitoring.

Microchip atmospheric pressure chemical ionization source for mass spectrometry.

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD < 10%) and linearity (r(2) > 0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 muL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector.

Atmospheric pressure photoionization-mass spectrometry with a microchip heated nebulizer.

A novel, microfabricated heated nebulizer chip for atmospheric pressure photoionization-mass spectrometry (APPI-MS) is presented. The chip consists of fluidic and gas inlets, a mixer, and a nozzle etched onto silicon wafer that is anodically bonded to a Pyrex glass wafer, on which an aluminum heater is sputtered. A krypton discharge lamp is used as the source for 10-eV photons to initiate the photoionization process. Dopant, delivered as part of the sample solution, is used to achieve efficient ionization. The use of the microfabricated heated nebulizer with APPI in the analysis of four analytes is demonstrated, and the spectra are compared to those obtained with a conventional APPI source. Ionization in positive and negative ion modes was successfully achieved and the spectra were mainly similar to those obtained with conventional APPI, indicating that the ionization in microfabricated and conventional APPI sources takes place by the same mechanisms. The flow rates with conventional APPI are approximately 100 muL/min, whereas the microchip heated nebulizer allows the use of flow rates 0.05-5 muL/min, thus being compatible with microfluidic separation systems or micro- and nano-LC. A stable signal was demonstrated throughout a 5-h measurement, which proved the excellent stability of the micro-APPI. The same heated nebulizer chip can be used for weeks.